The present invention relates to influenza virus vaccine compositions and methods of treating or preventing influenza infection and disease in mammals. Influenza is caused by an RNA virus of the myxovirus group. Influenza viruses can be classified into three types (A, B and C), based on antigenic differences in the nucleoprotein and the matrix protein. Type A, which includes several subtypes, causes widespread epidemics and global pandemics. Type B causes regional epidemics. Influenza C is less severe and has been isolated from humans and pigs. Type C causes sporadic cases and minor, local outbreaks. Influenza A viruses can be further classified based on the viral surface proteins hemagglutinin (HA or H) and neuraminidase (NA or N). There are sixteen known H subtypes and nine known N subtypes of Type A viruses; while there is only one known H subtype and one N subtype of Type B viruses. Typical nomenclature identifies an influenza virus by both proteins, e.g., H3N2.
Type A and B influenza viruses each contain 8 RNA segments, while type C only has 7 RNA segments. Influenza A is most important and is very pathogenic for man, as well as for animals, for example pigs and horses. Type B influenza causes disease in humans. These virus types are distinguished in part on the basis of differences in two structural proteins, the nucleoprotein, found in the center of the virus, and the matrix protein, which forms the viral shell. The virus is transmitted through the air, mainly in droplets expelled during coughing and sneezing. The influenza viruses cause an infection of the respiratory tract, which is usually accompanied with coughing, high fever and myalgia.
Although an influenza infection does not often lead to the death of the infected individual, the morbidity can be severe. As a consequence thereof influenza epidemics may lead to substantial economic loss. Furthermore, influenza infection can be more dangerous for certain groups of individuals, such as those having suffered from a heart attack, CARA patients or the elderly. A vaccine against influenza is therefore highly desirable.
Influenza Epidemiology and Virology
Pandemics of influenza A viruses continue to occur at sporadic intervals in human populations. Three have occurred in the twentieth century alone in 1918, 1957 and 19686-8. These worldwide pandemics are noted for their high mortality with rates approaching 30-50%9. For example, it is estimated that 20-40 million people died in the 1918 pandemic and at least 1.5 million people in the 1957 and 1968 outbreaks combined10. Whether a pandemic occurs from an act of nature or from the deliberate release of a novel influenza strain with pandemic potential, the extent of world travel will ensure the rapid global spread of the pandemic agent. Such an event could result in world-wide deaths totaling in the millions and severely impact health care systems such that economies and governments of smaller countries could collapse9,11.
The capacity of the influenza virus to cause disease in a recurring manner is due to a complex set of factors that include: 1) the presence of an established reservoir of influenza A viruses of different subtypes in shorebirds and waterfowl; 2) the ability of avian influenza viruses to recombine with influenza viruses of other animals, most notably swine12, a process termed ‘antigenic shift’; 3) accumulation of mutations in viral gene products caused by a lack of proofreading activity of the viral RNA polymerase, a process termed ‘antigenic drift’. These reassortment and mutation events combine to cause the well-characterized antigenic variability in the two surface glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA)13-15 which provides the virus a mechanism for escaping immune responses, particularly neutralizing antibodies, induced as the result of previous infections or vaccinations. Antigenic shift, which occurs only among influenza A viruses, results in major antigenic change introducing viruses with a new gene segment(s). Antigenic shift can occur when an animal influenza A virus is transmitted directly to humans, such as the transmission of the H1N1 from swine-to-human16 or the transmission of the H5N1, H7N7 or H9N2 variants from avian to human17,18. Alternatively, a virus may acquire a new gene segment(s) as a result of genetic reassortment between animal and human influenza A viruses, the cause of the 1957 H2N2 and 1968 H3N2 pandemics19.
Since 1997, several novel avian subtypes have crossed the so-called species barrier from domestic poultry to humans and have caused a spectrum of mild to severe and even fatal human disease. In 1997, 18 cases of human infection with highly pathogenic avian H5N1 influenza viruses, including 6 deaths were documented in Hong Kong following outbreaks of disease in domestic poultry. Avian H5N1 viruses reemerged in Hong Kong and from Dec. 30, 2003 to Mar. 17, 2004, there were 12 human cases of confirmed H5N1 influenza in Thailand and 23 in Vietnam, including 23 deaths. As of May 2006, approximately 115 deaths have been attributed to H5N1 infection. The H5N1 strain does not jump easily from birds to humans or between humans. However since the human virus, H3N2, can coexist with avian influenza viruses and is widespread in pigs from southeast China, reassortment has the potential to occur with a highly pathogenic human-to-human transmissible H5N1 being the result. Although these wholly avian viruses were associated with only limited human-to-human transmission, their repeated emergence in humans highlights the potential for the generation of an avian-human reassortant virus with the potential for spread in the human population. Thus, the development of effective vaccines against these avian subtypes is of the highest public health priority.
Vaccine production must rely on surveillance programs to predict the influenza subtypes likely to have global impact on human health. The time required to produce subtype-matched vaccines, composed of inactivated or ‘split’ virions, typically requires a minimum of 6-8 months. In the face of a serious influenza virus pandemic caused by a viral subtype, this lag time could allow for national or international spread with excessive morbidity and mortality.
Virus Structures
An influenza virus is roughly spherical, but it can also be elongated or irregularly shaped. Inside the virus, eight segments of single-stranded RNA contain the genetic instructions for making the virus. The most striking feature of the virus is a layer of spikes projecting outward over its surface. There are two different types of spikes: one is composed of the molecule hemagglutinin (HA), the other of neuraminidase (NA). The HA molecule allows the virus to “stick” to a cell, initiating infection. The NA molecule allows newly formed viruses to exit their host cell without sticking to the cell surface or to each other. The viral capsid is comprised of viral ribonucleic acid and several so called “internal” proteins (polymerases (PB1, PB2, and PA, matrix protein (M1) and nucleoprotein (NP)). Because antibodies against HA and NA have traditionally proved the most effective in fighting infection, much research has focused on the structure, function, and genetic variation of those molecules. Researchers are also interested in two non-structural proteins M2 and NS1; both molecules play important roles in viral infection.
Type A subtypes are described by a nomenclature system that includes the geographic site of discovery, a lab identification number, the year of discovery, and in parentheses the type of HA and NA it possesses, for example, A/Hong Kong/156/97 (H5N1). If the virus infects non-humans, the host species is included before the geographical site, as in A/Chicken/Hong Kong/G9/97 (H9N2).
Virions contain 7 segments (influenza C virus) to 8 segments (influenza A and B virus) of linear negative-sense single stranded RNA. Most of the segments of the virus genome code for a single protein. For many influenza viruses, the whole genome is now known. Genetic reassortment of the virus results from intermixing of the parental gene segments in the progeny of the viruses when a cell is co-infected by two different viruses of a given type. This phenomenon is facilitated by the segmental nature of the genome of influenza virus. Genetic reassortment is manifested as sudden changes in the viral surface antigens.
Antigenic changes in HA and NA allow the influenza virus to have tremendous variability. Antigenic drift is the term used to indicate minor antigenic variations in HA and NA of the influenza virus from the original parent virus, while major changes in HA and NA which make the new virions significantly different, are called Antigenic shift. The difference between the two phenomena is a matter of degree.
Antigenic drift (minor changes) occurs due to accumulation of point mutations in the gene which results in changes in the amino acids in the proteins. Changes which are extreme, and drastic (too drastic to be explained by mutation alone) result in antigenic shift of the virus. The segmented genomes of the influenza viruses reassort readily in double infected cells. Genetic reassortment between human and non-human influenza virus has been suggested as a mechanism for antigenic shift. Influenza is a zoonotic disease, and an important pathogen in a number of animal species, including swine, horses, and birds, both wild and domestic. Influenza viruses are transferred to humans from other species.
Because of antigenic shift and antigenic drift, immunity to an influenza virus carrying a particular HA and/or NA protein does not necessarily confer protective immunity against influenza virus strains carrying variant, or different HA and/or NA proteins. Because antibodies against HA and NA have traditionally proved the most effective in fighting influenza virus infection, much research has focused on the structure, function and genetic variation of those molecules.
Role of Cellular Immune Responses in Protection Against Influenza
Cellular immune responses are known to contribute to the control of viral replication in vivo and to mediate viral clearance. In murine models, influenza-specific CD8+ cytotoxic T-lymphocytes (CTL) limit virus replication and protect against lethal virus challenge20-27. Recovery from infection correlated with virus-specific CD8+ CTL activity22 and lack of CD8+ CTL activity was associated with delayed viral clearance and increased mortality28. Studies completed by Ulmer and Okuda using a DNA vaccine encoding the viral nucleoprotein and M gene proteins, respectively are particularly relevant. These vaccines induced influenza-specific CD8+ CTL that provided cross-strain protection27,29,30. The contribution of CTL and Helper T-lymphocytes (HTL) was definitively demonstrated by adoptive transfer of CD8+ and CD4+ T-lymphocytes31. Similarly, Epstein and colleagues demonstrated that either CD8+ or CD4+ T-lymphocytes promoted survival in mice immunized with an experimental DNA vaccine encoding internal viral proteins32. Finally, virus specific HTL augment the generation of CTL and size of the CTL memory pool, an effect known to be associated with long term protection33. Cellular immune responses clearly contribute to the control and clearance of infection and reduce pathogenesis.
The exposure to an influenza virus of one subtype often induces immune responses that protect against infection or disease with another subtype, a phenomena referred to as Heterosubtypic Immunity (HSI)34-37. The mechanisms of heterosubtypic immunity appears to involve functional activity of both CD8+ and CD4+ T-lymphocytes23,26,38-41, although more recently antibody responses have also been implicated42. HSI is not only observed using the murine models; influenza virus-specific CTL appear to provide partial protection against multiple influenza A virus strains in humans. Early human studies demonstrated that cellular immune responses play a role in controlling influenza infection43,44. McMichael and colleagues inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/l/79 virus and evaluated the protective effects of serum antibody and cytotoxic T-cell immunity against influenza.43 It was found that all subjects with demonstrable T-cell responses cleared virus effectively. Sonoguchi and colleagues found that students previously infected with H3N2 virus were partially protected against subsequent infection with H1N1 subtype virus suggesting cross-subtype protection in humans during sequential epidemics. Thus, the use of vaccines to induce cellular responses against pandemic influenza virus is logical and the development of suitable vaccine technologies is warranted.
Immune system-mediated selection pressure on influenza virus can lead to CTL viral escape mutants45-47. While this phenomena clearly documents the importance of virus-specific CTL it also reveals a potential limitation for vaccines designed to induce CTL responses. However, the use of carefully selected epitopes in the design of a vaccine provides a means to address this problem. Selection of epitopes that are highly conserved amongst multiple viral strains is the first step and the selection of those epitopes predicted to be capable of inducing CTL responses to the majority of related epitopes is the second step.
Role of Humoral Immune Responses in Protection Against Influenza
Influenza vaccines are formulated to include human influenza strains predicted to pose the greatest risk for infectious spread. This vaccine development process requires approximately 6-8 months using conventional strains. Neutralizing antibodies induced primarily to the surface hemagglutinin protein by the conventional vaccines are highly protective. However, due to antigenic drift of the virus, the vaccines must be reformulated on a yearly basis. The danger persists that a “new” strain will emerge by antigenic shift for which the human population has little or no pre-existing immunity. Also, since vaccine production relies on embryonated chicken eggs or potentially cells in tissue culture, there are no assurances that sufficient new virus can be produced even within the 6-8 month time frame especially if the new influenza strain is lethal to birds. Pandemic influenza vaccine development would benefit by inclusion of conserved B cell epitopes capable of inducing protective immune responses. To this end, it has been reported that the external domain of the transmembrane viral M2 protein is highly conserved and that antibodies directed to this epitope are protective in mice48-54. The M2 protein is an integral membrane protein of influenza A virus that is expressed at the plasma membrane in virus-infected cells. Due to the low abundance of the protein in the virus, the mechanism of protection of the antibody response directed against this epitope is not mediated via viral neutralization but rather by antibody-dependent, cell-mediated cytotoxicity51.
Conserved CTL, HTL and B-cell epitopes can be used as the basis for a vaccine designed to augment and improve prototype pandemic vaccine candidates that may be poorly immunogenic or a sub-optimal match against a pandemic strain that emerges. The advantages to using defined epitopes in vaccines are many but one advantage is that many epitopes can be incorporated into a vaccine to induce a broadly specific immune response targeting numerous viral gene products. Data from natural infection studies wherein human memory CTL specific to influenza A virus were restricted by multiple HLA Class I alleles have shown that responses within a given individual were broadly directed to epitopes within the NP, NA, HA, M1, NS1 and M2 viral proteins.
Design and Testing of Vaccines to Induce Cellular and Humoral Immune Responses:
The use of recombinant DNA technology to produce influenza vaccines offers several advantages: a recombinant DNA influenza vaccine can be produced under safer and more stringently controlled conditions; propagation with infectious influenza in eggs is not required; recombinant HA protein can be more highly purified, virtually eliminating side effects due to contaminating proteins; purification procedures for recombinant HA do not have to include virus inactivation or organic extraction of viral membrane components, therefore avoiding denaturation of antigens and additional safety concerns due to residual chemicals in the vaccine. Production of HA via recombinant DNA technology provides an opportunity to avoid the genetic heterogeneity which occurs during adaptation and passage through eggs, which should make it possible to better match vaccine stains with influenza epidemic strains, resulting in improved efficacy; and a recombinant approach may also allow for strain selection later in the year, thereby allowing time for selections based on more reliable epidemiological data.
A major obstacle to the development of vaccines that induce immune responses is the selection of a suitable delivery format. DNA plasmid vaccines and viral vectors, used either alone or together, and recombinant protein or peptides are logical vaccine delivery formats; however, each format has advantages and disadvantages. For example, DNA vaccines are readily produced and safe to administer but potency has been lacking, especially in clinical trials, requiring the administration of large (milligram) doses59-65. Studies completed in small animals have indicated increased vaccine potency66-69. Polymer formulation technology based on polyvinylpyrrolidone (PVP) can also be utilized. PVP is a nontoxic formulation excipient used to enhance DNA plasmid uptake by muscle cells70-73. Such vaccine design parameters can correct for at least some of the limitations of naked-DNA vaccine technology.
The use of viral vectors to deliver vaccines has raised concerns, usually related to safety and pre-existing immunity to the vector. However, AlphaVax replicons are reported to be safe, non-transmissible and there is a general lack of pre-existing immunity to the vector. Another delivery vehicle that is being evaluated is peptides in adjuvant. Generally, peptides in adjuvant have shown to be immunogenic and efficacious in humans. However, there are concerns regarding vaccine formulation wherein high numbers of peptides will need to be delivered.
Several adjuvants have been developed for the administration of influenza virus vaccines, including alum based compounds, emulsions (e.g. MF59), (lipophilic immune stimulating complexes ISCOMS) containing Quil A adjuvant) and liposomes. A development of the liposomal technique has been the use of immunopotentiating reconstituted influenza virosomes (IRIVs) as antigen delivery systems. See Mischler, R. and Metcalfe, I. C., Vaccine 20: B17-B23 (2002). The IRIV vaccine delivery system is comprised of spherical unilamellar vesicles comprising naturally occurring phospholipids (PL) and phosphatidylcholine (PC) and envelope phospholipids originating from influenza virus used to provide influenza virus NA and HA glycoproteins. See id. The fusion mechanism of IRIVs enables stimulation of the MHC Class I or Class II pathway, depending upon how antigens are presented to the APCs. Virosomes are able to induce either a B- or T-cell response. See id.
The use of smaller polypeptides comprising antigenic epitopes in vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. There is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations. The epitopes for inclusion in an epitope-based vaccine may be selected from conserved regions of influenza antigens, which thereby reduces the likelihood of escape mutants. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.
An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes (e.g., multiple HTL epitope epitopes), and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.
Several groups have established the mouse model as a tool for evaluating the efficacy of influenza vaccines26-31,74,75. The testing of vaccines comprised of epitopes restricted by HLA is a unique challenge, requiring the appropriate restriction elements. Specifically cell-surface expressed HLA Class II molecules for HTL epitopes on antigen presenting cells are required. With regard to evaluating Class II-restricted responses, HLA-DR4 mice are available commercially. Most HTL epitopes restricted to HLA Class II can bind murine H-2 IAb molecules and initiate a response80.
Virus-specific, human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL) are known to play a major role in the prevention and clearance of virus infections in vivo (Oldstone, et al., Nature 321:239, 1989; Jamieson, et al., J. Virol. 61:3930, 1987; Yap, et al., Nature 273:238, 1978; Lukacher, et al., J. Exp. Med. 160:814, 1994; McMichael, et al., N. Engl. J. Med. 309:13, 1983; Sethi, et al., J. Gen. Virol. 64:443, 1983; Watari, et al., J. Exp. Med. 165:459, 1987; Yasukawa, et al., J. Immunol. 143:2051, 1989; Tigges, et al., J. Virol. 66:1622, 1993; Reddenhase, et al., J. Virol. 55:263, 1985; Quinnan, et al., N. Engl. J. Med. 307:6, 1982). HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.
Virus-specific T helper lymphocytes are also known to be critical for maintaining effective immunity in chronic viral infections. Historically, HTL responses were viewed as primarily supporting the expansion of specific CTL and B cell populations; however, more recent data indicate that HTL may directly contribute to the control of virus replication. For example, a decline in CD4+ T cells and a corresponding loss in HTL function characterize infection with HIV (Lane, et al., N. Engl. J. Med. 313:79, 1985). Furthermore, studies in HIV infected patients have also shown that there is an inverse relationship between virus-specific HTL responses and viral load, suggesting that HTL plays a role in controlling viremia (see, e.g., Rosenberg, et al., Science 278:1447, 1997).
The epitope approach, as we describe herein, allows the incorporation of various antibody, CTL and HTL epitopes, from various proteins, in a single vaccine composition. Such a composition may simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.
The technology relevant to multi-epitope (“minigene”) vaccines is developing. Several independent studies have established that induction of simultaneous immune responses against multiple epitopes can be achieved. For example, responses against a large number of T cell specificities can be induced and detected. In natural situations, Doolan, et al. (Immunity, Vol. 7(1):97-112 (1997)) simultaneously detected recall T cell responses, against as many as 17 different P. falciparum epitopes using PBMC from a single donor. Similarly, Bertoni and colleagues (J. Clin. Invest., 100(3):503-13 (1997)) detected simultaneous CTL responses against 12 different HBV-derived epitopes in a single donor. In terms of immunization with multi-epitope nucleic acid vaccines, several examples have been reported where multiple T cell responses were induced. For example, minigene vaccines composed of approximately ten MHC Class I epitopes in which all epitopes were immunogenic and/or antigenic have been reported. Specifically, minigene vaccines composed of 9 EBV (Thomson, et al., Proc. Natl. Acad. Sci. USA, 92(13):5845-49 (1995)), 7 HIV (Woodberry, et al., J. Virol., 73(7):5320-25 (1999)), 10 murine (Thomson, et al., J. Immunol., 160(4):1717-23 (1998)) and 10 tumor-derived (Mateo, et al., J. Immunol., 163(7):4058-63 (1999)) epitopes have been shown to be active. It has also been shown that a multi-epitope DNA plasmid encoding nine different HLA-A2.1- and A11-restricted epitopes derived from HBV and HIV induced CTL against all epitopes (Ishioka, et al., J. Immunol., 162(7):3915-25 (1999)).
Recently, several multi-epitope DNA plasmid vaccines specific for HIV have entered clinical trials (Nanke, et al., Nature Med., 6:951-55 (2000); Wilson, C. C., et al., J. Immunol. 171(10):5611-23 (2003).
Thus, vaccines containing multiple MHC Class I and Class II (i.e., HTL) epitopes can be designed, and presentation and recognition can be obtained for all epitopes. However, the immunogenicity of such multi-epitope constructs appears to be strongly influenced by a number of variables, a number of which have heretofore been unknown. For example, the immunogenicity (or antigenicity) of the same epitope expressed in the context of different vaccine constructs can vary over several orders of magnitude. Thus, there exists a need to identify strategies to optimize such multi-epitope containing vaccine constructs. Such optimization is important in terms of induction of potent immune responses and ultimately, for clinical efficacy. Accordingly, the present invention provides strategies to optimize antigenicity and immunogenicity of multi-epitope vaccines encompassing a certain number of epitopes. The present invention also provides optimized multi-epitope containing vaccines, particularly minigene vaccines, generated in accordance with these strategies.